Targeting ALK averts ribonuclease 1-induced immunosuppression and enhances antitumor immunity in hepatocellular carcinoma

Tumor-secreted factors contribute to the development of a microenvironment that facilitates the escape of cancer cells from immunotherapy. In this study, we conduct a retrospective comparison of the proteins secreted by hepatocellular carcinoma (HCC) cells in responders and non-responders among a cohort of ten patients who received Nivolumab (anti-PD-1 antibody). Our findings indicate that non-responders have a high abundance of secreted RNase1, which is associated with a poor prognosis in various cancer types. Furthermore, mice implanted with HCC cells that overexpress RNase1 exhibit immunosuppressive tumor microenvironments and diminished response to anti-PD-1 therapy. RNase1 induces the polarization of macrophages towards a tumor growth-promoting phenotype through activation of the anaplastic lymphoma kinase (ALK) signaling pathway. Targeting the RNase1/ALK axis reprograms the macrophage polarization, with increased CD8+ T- and Th1- cell recruitment. Moreover, simultaneous targeting of the checkpoint protein PD-1 unleashes cytotoxic CD8+ T-cell responses. Treatment utilizing both an ALK inhibitor and an anti-PD-1 antibody exhibits enhanced tumor regression and facilitates long-term immunity. Our study elucidates the role of RNase1 in mediating tumor resistance to immunotherapy and reveals an RNase1-mediated immunosuppressive tumor microenvironment, highlighting the potential of targeting RNase1 as a promising strategy for cancer immunotherapy in HCC.

Editorial Note: This manuscript has been previously reviewed at another journal that is not operafing a transparent peer review scheme.This document only contains reviewer comments and rebuftal lefters for versions considered at Nature Communicafions.

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): We appreciate the responses to the prior comments and revisions that the authors have provided.We think that these have significantly strengthened the paper.We have the following comments related to the paper: Major 105 (Figure 1a-d) We and another reviewer have expressed concern about using this extremely limited dataset as a scienfific jusfificafion for pursuing RNAse1 on a cohort of 10 pafients with the previously menfioned confounding issues present in this cohort.If the authors would simply like to explain that they have studied RNase1 and other RNAses in the past in the context of other cancers and found the overall survival associafions in large cohorts of data intriguing for invesfigafion in combinafion with this (exceedingly limited) responder/non-responder data, this would be acceptable.
The strong desire to showcase this dataset of PD1 monotherapy-treated pafients has significant limitafions when there are unquesfionably inadequate data.While I do appreciate that a data set of pafients treated exclusively with PD-1 monotherapy is unique, this doesn't change the limitafions of a 10 pafient study.Pafients with HCC treated exclusively with immunotherapy are also not rare-Trem/Durva is a standard of care first line regimen by NCCN guidelines.Thousands of pafients are receiving this as first line treatment for HCC now.Finally, one could argue that the value of this cohort is further reduced as excepfionally few pafients receive PD1 monotherapy-there is not a clinical need to improve a regimen that is not being used in pafients.To be clear, we do agree that this data is useful and important, but simply cannot be used as a jusfificafion for the pursuit of RNase1.The value it brings does not outweigh its severe limitafions.
We acknowledge the IHC analysis of 13 pafients that is included from a separate cohort, but again, this seems supplementary rather than foundafional in jusfifying this study.493 / Discussion: Seeing as the authors have published a paper (PMCID: PMC10321278) demonstrafing that RNase1 enhances anfi-tumor immunity in a syngeneic model of breast cancer they developed, it would be appropriate to discuss possible reasons for the opposing phenotypes in these models.We appreciate the responses to the prior comments and revisions that the authors have provided.We think that these have significantly strengthened the paper.We have the following comments related to the paper: Authors' Response: We thank the reviewer for the positive comments.For the additional questions, we have answered as follow: Major 105 (Figure 1a-d) We and another reviewer have expressed concern about using this extremely limited dataset as a scientific justification for pursuing RNAse1 on a cohort of 10 patients with the previously mentioned confounding issues present in this cohort.If the authors would simply like to explain that they have studied RNase1 and other RNAses in the past in the context of other cancers and found the overall survival associations in large cohorts of data intriguing for investigation in combination with this (exceedingly limited) responder/non-responder data, this would be acceptable.
The strong desire to showcase this dataset of PD1 monotherapy-treated patients has significant limitations when there are unquestionably inadequate data.While I do appreciate that a data set of patients treated exclusively with PD-1 monotherapy is unique, this doesn't change the limitations of a 10 patient study.Patients with HCC treated exclusively with immunotherapy are also not rare-Trem/Durva is a standard of care first line regimen by NCCN guidelines.Thousands of patients are receiving this as first line treatment for HCC now.Finally, one could argue that the value of this cohort is further reduced as exceptionally few patients receive PD1 monotherapy-there is not a clinical need to improve a regimen that is not being used in patients.To be clear, we do agree that this data is useful and important, but simply cannot be used as a justification for the pursuit of RNase1.The value it brings does not outweigh its severe limitations.
We acknowledge the IHC analysis of 13 patients that is included from a separate cohort, but again, this seems supplementary rather than foundational in justifying this study.
Authors' Response: We thank the reviewer for the comments.We agreed with the reviewer that more patient cases will further ensure the role of RNase1 in immunotherapy.In our story, RNase1 was first identified as a potential factor that was associated with ICI response in two small independent cohorts, and then we used multiple criteria to support the notion, including RNase1-overexpressing and knockout HCC orthotopic mouse model and patient tissue microarray analyses.These pre-clinical studies, tissue microarray data plus the two independent small cohorts pointing out the potential importance for RNase1 as a resistant marker in the future.We understand the cohort size is limited, but that is not the only data present in the story.We will add the following to point out the limitation of small cohort in discussion as following."We understand the size of cohort is small, but the results are encouraging, and potentially important therefore we performed the following laboratory experiments to provide additional support on the concept." We also agreed with the reviewer that Durva/Treme (anti-PD-L1 + anti-CTLA-4 combination immunotherapy) has been approved as a standard of care by NCCN guidelines.However, the roles of CTLA-4 and PD-1 in inhibiting immune responses, including antitumor responses, are largely distinct.Thus, the potential mechanisms associated with monotherapies and combined immunotherapy are quite different.And although few patients receive anti-PD1 monotherapy because the lack of survival benefit, there is clinical need to exploring the resistant mechanism to improve and recommend biomarker-guided combination therapies.
We have added the explanation in the manuscript as follow: "Considering that RNase1 and other RNases were significantly associated with overall survival in large patient cohorts in different cancer types, RNase 1 was the top candidate for further investigation due to its upregulation in non-responders receiving Nivolumab and its unknown function in tumor immunity." 493 / Discussion: Seeing as the authors have published a paper (PMCID: PMC10321278) demonstrating that RNase1 enhances anti-tumor immunity in a syngeneic model of breast cancer they developed, it would be appropriate to discuss possible reasons for the opposing phenotypes in these models.
Authors' Response: We thank the reviewer for the suggestion.We have added the discussion in manuscript as follow: "Interestingly, recent results of Wang et al., who found that RNase1 elicits adaptive immune response against breast cancer by boosting CD4 + T cell activation through associating with EphA4 (PMCID: PMC10321278).The different binding receptors of RNase1 on macrophage and CD4 + T cells might cause the differential responses in the two cell and cancer types.As we know, the number of TAMs in the TME accounts for 20% to 40% among all HCC infiltrating lymphocytes and even more in some rare HCC subtypes (PMID: 36792608).Thus, RNase1 exhibits immunosuppressive effects in our HCC mouse models through activating ALK on TAMs.RNase1 may regulate CD4 + T cells activation through binding to different receptor, EPHA4, thus it would be worthwhile to evaluate the proportion of TAMs and CD4 + TILs, as well as the expression level of ALK and EphA4 in different patients when these markers are used for choosing appropriate treatment strategy."Authors' Response: We thank the reviewer for the comments, and we have edited the description as follow: "Multiple strategies have been approved for first or second-line treatment for advanced HCC, such as sorafenib, cabozantinib, lenvatinib, atezolizumab plus bevacizumab, tremelimumab plus durvalumab, and others."57-60: This is also outdated.See attached hepatocellular carcinoma clinical guidelines.
Authors' Response: We thank the reviewer for the suggestion.We have modified the description to emphasize the lack of biomarker dilutes the efficacy of current combinational therapies.
"Although several novel combinations are in development, including anti-PD-1/PD-L1 antibodies in combination with anti-CTLA4 and/or anti-VEGF antibodies or multikinase inhibitors, the lack of predictive biomarkers to guide the use of those combinational strategies dilute the efficacy."

Minor 47 -
48 Sorafenib and Lenvafinib are not the preferred first line recommendafions by the NCCN for advanced HCC.In the US, pafients do not receive these unless there are contraindicafions to immunotherapy.Standard of care preferred first line therapies are atezolizumab+bevacizumab or tremelimumab+durvalumab. Second line treatment opfions include Lenvafinib, sorafenib, cabozanfinib, regorafenib, and others.57-60: This is also outdated.See aftached hepatocellular carcinoma clinical guidelines.100: gene signatures of monocyte to M2-macrophage 116 / Figure 1e/Supp

Table 3 :
I don't see where the numeric overall survival is stated.
Minor 47-48 Sorafenib and Lenvatinib are not the preferred first line recommendations by the NCCN for advanced HCC.In the US, patients do not receive these unless there are contraindications to immunotherapy.Standard of care preferred first line therapies are atezolizumab+bevacizumab or tremelimumab+durvalumab. Second line treatment options include Lenvatinib, sorafenib, cabozantinib, regorafenib, and others.